LeMDR和LeMRP调控紫草天然产物跨质膜和液泡膜转运的功能及机制解析

The cells of boraginaceae plants, such as Lithospermun erythrorhizon and Onosma paniiculatum, can biosynthesize important natural products (shikonin and its derivatives) with significant medicinal values. These compounds can be transported out of the cells or partly stored in the vacuole across the plasma membrane and tonoplast, respectively, and the transport efficiency has important feedback effect on regulating their formation. Although previous studies showed that ABC-type transporters play critical roles in the transport of natural products across the membrane, it still remains unclear about the issue that whether ABC transporters are needed for the transport of liposoluble naphthoquinone compounds -shikonin and its derivatives, or the intermediate products for their biosynthesis. In my previous NSFC project, we discovered two ABC-type genes, LeMDR and LeMRP, and the pre-experimental results suggest that theses two genes possibly play pivotal roles in the transport of shikonin and its derivatives across the plasma membrane and tonoplast, respectively. In this project, we aim to clone the full-length cDNA of these two genes, and coordinately dissect their functions and transport mechanisms by analyzing the correlation between the gene and protein expression patterns and the biosynthesis and transport of the compounds; analyzing the effect of core site mutation on the protein activity and inhibitor regulation; analyzing the transport characterizations with the gene functional complementation in yeast ABC mutants or with the xenopus oocyte system; analyzing the compounds biosynthesis and membrane vesicle transport characterizations via the transgenic strategies (Overexpression, RNA interference or CRISPR/Cas9) and so on. The results of this project will not only lay foundation for further revealing the transport mechanism of liposoluble compounds and efficiently regulating their biosynthesis by focusing on the transport point, but also provide key target genes for bioengineering practice.

紫草细胞可合成具有重要药用价值的天然产物-紫草宁及其衍生物，并通过跨膜转运到胞外或部分储存于液泡中，而转运效率对于其高效合成又起到重要的反馈调控作用。尽管ABC类转运蛋白在天然产物跨膜转运中起重要作用，但对于亲脂性的紫草宁及其衍生物这种萘醌类分子或合成过程中的中间产物的跨膜转运是否需要ABC蛋白的参与还很不清楚。申请人在结题的NSFC项目研究中发掘到2个ABC类基因LeMDR和LeMRP，预研结果表明它们极有可能分别在紫草天然产物跨质膜和液泡膜转运中起重要作用。本项目拟克隆其全长，并通过基因和蛋白表达与物质合成及转运的相关性分析、蛋白核心位点突变与抑制剂活性调控分析、酵母突变体互补转运或爪蟾卵母细胞转运特征分析、紫草转基因（OE、RNAi或CRISPR/Cas9）物质合成及膜囊泡制备转运特征分析等协同解析其功能和机制，为深入揭示亲脂性天然产物的跨膜转运及靶向转运环节来高效调控其生物合成奠定基础并提供关键靶标。

紫草细胞可合成具有重要药用价值的天然产物-紫草宁及其衍生物，并通过跨膜转运到胞外或部分可能储存于液泡中，而转运效率对于其高效合成又可能起到重要的反馈调控作用。尽管ABC类转运蛋白在天然产物跨膜转运中起重要作用，但对于亲脂性的紫草宁及其衍生物这种萘醌类分子的跨膜转运是否需要ABC蛋白的参与还很不清楚。本项目研究结果为深入揭示紫草宁合成调控及转运的分子机制，以及非模式植物的次生代谢生物工程实践奠定了基础。主要结果概述如下：. （1）克隆了硬紫草ABC转运蛋白基因家族的关键成员LeMDR和LeMRP的cDNA全长和启动子，并分析了其分子特征；（2）通过酵母突变体的异源表达功能分析体系解析了LeMDR和LeMRP 对紫草宁的跨膜转运功能；（3）成功地获得了LeMDR和LeMRP基因的过表达和RNA干扰转基因毛状根株系；（4）通过对转基因毛状根的色素和基因表达分析明确了LeMDR和LeMRP基因对紫草宁及其衍生物的合成可起到间接的正反馈调控作用；（5）通过LeMDR功能域突变的酵母突变体异源表达功能分析体系阐明了其紫草宁转运关键功能域；（6）通过激素及培养基转换处理的愈伤组织和幼苗不同部位等的qPCR分析，阐明了LeMDR和LeMRP基因的时空表达模式；（7）基于全基因组分析，鉴定了硬紫草全部ABC转运蛋白家族成员，阐明了其进化关系；（8）获得了过表达LeMDR和LeMRP的各一个高产紫草宁及其衍生物的转基因毛状根株系，可应用于生产实践。